Scansite 2.0 is a computational tool that predicts cell signaling interactions using short sequence motifs. It identifies motifs recognized by modular signaling domains, such as SH2, SH3, PDZ, 14-3-3, and PTB domains, and their binding or substrate specificities. These motifs are represented as position-specific scoring matrices (PSSMs) derived from experimental data. Scansite 2.0 allows users to search protein databases for motif matches and construct biological pathways in silico. It includes 62 motifs for various kinase and binding domains, with significant improvements in performance and user interface. The tool supports restricted searches, isoelectric point and molecular weight sorting, and user-defined motifs. It also includes new programs for sequence matching and non-quantitative motif searches. Scansite 2.0 is available online at http://scansite.mit.edu. It uses a relational database for faster searches and allows users to submit their own motifs for inclusion in the database. The tool is open-source under the GNU General Public License, but PSSMs remain proprietary. Scansite 2.0 improves the speed and efficiency of protein interaction predictions, enabling targeted searches and integration with experimental data. Future developments include specialized databases, tissue-specific searches, and automated correlation with published data. The tool is valuable for functional annotation of protein interactions and identifying potential signaling pathways.Scansite 2.0 is a computational tool that predicts cell signaling interactions using short sequence motifs. It identifies motifs recognized by modular signaling domains, such as SH2, SH3, PDZ, 14-3-3, and PTB domains, and their binding or substrate specificities. These motifs are represented as position-specific scoring matrices (PSSMs) derived from experimental data. Scansite 2.0 allows users to search protein databases for motif matches and construct biological pathways in silico. It includes 62 motifs for various kinase and binding domains, with significant improvements in performance and user interface. The tool supports restricted searches, isoelectric point and molecular weight sorting, and user-defined motifs. It also includes new programs for sequence matching and non-quantitative motif searches. Scansite 2.0 is available online at http://scansite.mit.edu. It uses a relational database for faster searches and allows users to submit their own motifs for inclusion in the database. The tool is open-source under the GNU General Public License, but PSSMs remain proprietary. Scansite 2.0 improves the speed and efficiency of protein interaction predictions, enabling targeted searches and integration with experimental data. Future developments include specialized databases, tissue-specific searches, and automated correlation with published data. The tool is valuable for functional annotation of protein interactions and identifying potential signaling pathways.